Synthesis and biological study of aminoglycoside derivatives to overcome bacterial resistance

Aminoglycosides are broad-spectrum antibiotics that target the A-site of bacterial 16S ribosomal RNA. Their use, however, is increasingly threatened by the rapid spread of resistance. One of the most common determinants of aminoglycoside resistance in bacteria is the expression of a class of enzymes known as aminoglycoside 6′-N-acetyltransferases (AAC(6′)s). These enzymes use acetylcoenzyme A (AcCoA) to acetylate most aminoglycosides at the 6′-NH2, thus decreasing their affinity for RNA and leading to bacterial resistance. One strategy pursued by the Auclair research group to overcome aminoglycoside resistance is to develop AAC(6′) inhibitors. Chapter 2 of this thesis describes enzymatic studies with the group's first generation of inhibitors, aminoglycoside-CoA bisubstrates and truncated analogs. The bisubstrates exhibited potent nanomolar competitive inhibition of the Enterococcus faecium isoform AAC(6′)-Ii and proved to be useful mechanistic and structural probes. They did not however show activity in cells. Enzymatic studies with truncated bisubstrates allowed extensive SAR studies and led to the discovery of a second generation of inhibitors, one of which is capable of blocking aminoglycoside resistance in cells expressing AAC(6′)-Ii. To improve the potency of this compound, a series of derivatives with various amide groups replacing the ester functionality were synthesized. It was hypothesized that this modification would increase potency and biological stability. To our surprise however these small changes had a negative impact on the interaction between the inhibitors and AAC(6′)-Ii. Chapter 3 describes our second approach to counter aminoglycoside resistance, which involved the synthesis of new aminoglycosides designed to be active against resistant bacterial strains. A series of neamine N-6′-acylated derivatives were designed to retain the hydrogen bonding ability of 6′-N to RNA, yet disrupt binding to AAC(6′)-Ii. Some of these compounds showed moderate but not clinically relevant activity in cells.Finally, the third approach elaborated in Chapter 4 is to design prodrugs with dual, resistance inhibition and antibacterial activities. The goal was to develop aminoglycoside N-6′ derivatives as prodrugs that were expected to be extended to bisubstrate analogs by the CoA biosynthetic enzymes in bacteria. The resulting bisubstrates were expected to not only block aminoglycoside resistance via AAC(6′) inhibition, but to also kill bacteria by blocking the fatty acid biosynthetic pathway. A small series of aminoglycoside derivatives was thus synthesized. Unexpectedly none of them showed direct antibacterial activity. Preliminary biological studies however suggest that even in the absence of in vitro AAC(6′) inhibition, these molecules can potentiate the activity of aminoglycosides against an aminoglycoside resistant strain. To our knowledge they are the first reported prodrugs able to block aminoglycoside resistance in cells.

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